Folylpolyglutamate synthetase is an enzyme that metabolizes the naturally occurring folate cofactors and all classical folate antimetabolites to forms that contain 5-6 glutamic acid residues per molecule. These metabolites do not efflux from the cell, so that polyglutamation constitutes a mechanism to metabolically trap folates in the cell. When using antifolates in the treatment of cancer, the clinician is highjacking this mechanism to deliver enzyme inhibitors to the tumor cell and trap them there, in contact with their target for a substantial period of time. This application proposes to study the two control mechanisms that determine how much folylpolyglutamate synthetase activity exists in a cell. The mouse enzyme was shown to have a feedback by long chain polyglutamates that was adapted to the particularities of mouse folate metabolism. We propose to determine whether this exists with the human enzyme and the effects of such feedback on the pharmacology of the antifolates used in cancer, namely methotrexate, raltitredid and pemetrexed. The enzymology related to this putative feedback will also be explored. The control of expression of transcription of the fpgs gene will also be studied in normal human cells and leukemia cells. The fpgs gene is expressed in primitive hematopoietic cells, then silenced during differentiation. The mechanism of this silencing will be determined and the mechanism whereby leukemic cells circumvent this control will be determined. PUBLIC HEALTH RELEVANCE: The grant application proposes experiments that would lead to an understanding of how human folylpolyglutamate synthetase, an enzyme that limits the accumulation of folate compounds and analogs used for cancer therapy, carries out its functions. This enzyme determines whether individual human tumors are sensitive or insensitive to therapy with the antifolate drugs methotrexate, ralitrexid, and pemetrexed, and also the 5-fluoruracil- leucovorin combination. All of these drugs are currently used for treatment of human cancers, so any enhancement in our understanding of the enzyme that serves as a limitation for drug effects would help us design more rational cancer treatments with these drugs.